Document Detail


Site-directed cross-linking of b to the alpha, beta, and a subunits of the Escherichia coli ATP synthase.
MedLine Citation:
PMID:  10747904     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The b subunit dimer of the Escherichia coli ATP synthase, along with the delta subunit, is thought to act as a stator to hold the alpha(3)beta(3) hexamer stationary relative to the a subunit as the gammaepsilonc(9-12) complex rotates. Despite their essential nature, the contacts between b and the alpha, beta, and a subunits remain largely undefined. We have introduced cysteine residues individually at various positions within the wild type membrane-bound b subunit, or within b(24-156), a truncated, soluble version consisting only of the hydrophilic C-terminal domain. The introduced cysteine residues were modified with a photoactivatable cross-linking agent, and cross-linking to subunits of the F(1) sector or to complete F(1)F(0) was attempted. Cross-linking in both the full-length and truncated forms of b was obtained at positions 92 (to alpha and beta), and 109 and 110 (to alpha only). Mass spectrometric analysis of peptide fragments derived from the b(24-156)A92C cross-link revealed that cross-linking took place within the region of alpha between Ile-464 and Met-483. This result indicates that the b dimer interacts with the alpha subunit near a non-catalytic alpha/beta interface. A cysteine residue introduced in place of the highly conserved arginine at position 36 of the b subunit could be cross-linked to the a subunit of F(0) in membrane-bound ATP synthase, implying that at least 10 residues of the polar domain of b are adjacent to residues of a. Sites of cross-linking between b(24-156)A92C and beta as well as b(24-156)I109C and alpha are proposed based on the mass spectrometric data, and these sites are discussed in terms of the structure of b and its interactions with the rest of the complex.
Authors:
D T McLachlin; A M Coveny; S M Clark; S D Dunn
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  275     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  2000 Jun 
Date Detail:
Created Date:  2000-07-20     Completed Date:  2000-07-20     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  17571-7     Citation Subset:  IM    
Affiliation:
Department of Biochemistry, University of Western Ontario, London, Ontario, Canada N6A 5C1.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Substitution
Binding Sites
Cell Membrane / enzymology
Cross-Linking Reagents
Cysteine
Escherichia coli / enzymology*,  genetics
Macromolecular Substances
Models, Molecular
Mutagenesis, Site-Directed
Peptide Fragments / chemistry
Proton-Translocating ATPases / chemistry*,  genetics,  metabolism*
Recombinant Proteins / chemistry,  metabolism
Sequence Deletion
Chemical
Reg. No./Substance:
0/Cross-Linking Reagents; 0/Macromolecular Substances; 0/Peptide Fragments; 0/Recombinant Proteins; 52-90-4/Cysteine; EC 3.6.3.14/Proton-Translocating ATPases

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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